The organization of extracellular matrices by cells through the exertion of mechanical forces drives fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. Historically, our ability to investigate cell mechanical behavior has been limited by the technical challenges associated with measuring the sub-cellular pattern of cellular force generation and local matrix patterning in a 3-D environment. Thus our understanding of these fundamental processes is limited, especially in ocular tissues. Over the last several years, our research has been aimed at addressing these challenges through the development of new experimental models, use of emerging imaging technologies, and the application of quantitative analysis techniques. Research conducted in the prior grant period has provided important new insights into the roles of the small GTPases Rho and Rac in regulating the sub-cellular pattern of force generation and extracellular matrix reorganization by corneal fibroblasts within 3-D collagen matrices, as well as their response to local changes in mechanical stress. We now propose to further apply and expand our experimental models to address three key gaps in our understanding of corneal fibroblast mechanical behavior which have direct relevance to the process of corneal wound healing: 1) How do specific growth factors modulate the mechanical differentiation of corneal keratocytes?, 2) How do corneal keratocytes respond to large scale alterations in ECM mechanical properties?; and, 3) What regulates cell and matrix patterning during corneal keratocyte migration? To begin to answer these questions, we propose to: 1) perform a comprehensive assessment of how specific growth factors modulate keratocyte morphology, cytoskeletal organization, contractile force generation and matrix patterning within 3-D collagen matrices, and determine the role of Rho and Rac in mediated these effects, 2) investigate how alterations in matrix stiffness, density and anisotropy modulate the mechanical phenotype of corneal keratocytes following treatment with specific growth factors, and 3) Investigate how the interplay between Rho and Rac activation and alterations in ECM mechanical properties modulate cell and matrix patterning during corneal fibroblast migration within 3-D collagen matrices. Accomplishing these Specific Aims should provide important new insights into the underlying biochemical and biomechanical mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization in response to specific growth factors. This is fundamental information which can not be obtained using standard 2-D culture models. PUBLIC HEALTH RELVANCE: The mechanical behavior of corneal keratocytes plays a fundamental role in regulating the corneal response to lacerating injury or refractive surgery. We propose to use innovative 3-D culture models and quantitative imaging techniques to assess the mechanical response of keratocytes to biochemical and biophysical stimuli which have direct relevance to the process of corneal wound healing